Abstract:

The present invention provides polymeric delivery systems including
hindered ester moieties. Methods of making the polymeric delivery systems
and methods of treating mammals using the same are also disclosed.

Claims:

1. A compound of the Formula (I) ##STR00044## wherein:A is a capping group
or ##STR00045## R1 is a substantially non-antigenic water-soluble
polymer;L1 and L'1 are independently selected spacers having a
free electron pair positioned four to ten atoms from C(═Y1) or
C(═Y'1), preferably 4 to 8 and most preferably 4 to 5 atoms from
C(═Y1) or C(═Y'1);L2 and L'2 are
independently selected bifunctional linkers;Y1 and Y'1 are
independently O, S, or NR5;R2, R'2, R3, R'3 and
R5 are independently selected from the group consisting of hydrogen,
C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-19
branched alkyl, C3-8 cycloalkyl, C1-6 substituted alkyl,
C2-6 substituted alkenyl, C2-6 substituted alkynyl, C3-8
substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted
heteroaryl, C1-6 heteroalkyl, substituted C1-6 heteroalkyl,
C1-6 alkoxy, aryloxy, C1-6 heteroalkoxy, heteroaryloxy,
C2-6 alkanoyl, arylcarbonyl C2-6 alkoxycarbonyl,
aryloxycarbonyl, C2-6 alkanoyloxy, arylcarbonyloxy, C2-6
substituted alkanoyl, substituted arylcarbonyl, C2-6 substituted
alkanoyloxy, substituted aryloxycarbonyl, C2-6 substituted
alkanoyloxy and substituted arylcarbonyloxy, or R2 together with
R3 and R'2 together with R'3 independently form a
substituted or unsubstituted non-aromatic cyclohydrocarbon containing at
least three carbons;R4 and R'4 am independently selected from
the group consisting of OH, leaving groups, targeting groups, diagnostic
agents and biologically active moieties; and(p) and (p') are
independently zero or a positive integer, preferably zero or an integer
from about 1 to about 3, more preferably zero or 1;provided that R3
is a substituted or substituted hydrocarbon having at least three carbons
when R2 is H, and further provided that L1 is not the same as
C(R2)(R3).

3. The compound of claim 1, wherein R4 and R'4 are independently
selected from the group consisting of OH, methoxy, tert-butoxy,
para-nitrophenoxy and N-hydroxysuccinimidyl.

4. The compound of claim 1, wherein the biologically active moiety is
selected from the group consisting of --OH containing moieties and --SH
containing moieties.

5. The compound of claim 1, wherein the biologically active moiety is
selected from the group consisting of pharmaceutically active compounds,
enzymes, proteins, antibodies, monoclonal antibodies, single chain
antibodies and peptides.

14. The compound of claim 13, wherein the polyalkylene oxide is selected
from the group consisting of polyethylene glycol and polypropylene
glycol.

15. The compound of claim 13, wherein the polyalkylene oxide is selected
from the group consisting
of--Y71--(CH2CH2O)n--CH2CH2Y71--,--Y.s-
ub.71--(CH2CH2O)n--CH2C(═Y72)--Y71--,--Y-
71--C(═Y72)--(CH2).sub.a71--Y73--(CH2CH2-
O)n--CH2CH2--Y73--(CH2).sub.a71--C(═Y72)-
--Y71--,and--Y71--(CR71R72).sub.a72--Y73--(CH.sub-
.2).sub.b71--O--(CH2CH2O)n--(CH2).sub.b71--Y73--(-
CR71R72).sub.a72--Y71--,wherein:Y71 and Y73 are
independently O, S, SO, SO2, NR73 or a bond;Y72 is O, S,
or NR74;R71-74 are independently the sane moieties which can be
used for R2;(a71), (a72), and (b71) are independently zero or a
positive integer; and(n) is an integer from about 10 to about 2300.

16. The compound of claim 13, wherein the polyalkylene oxide is a
polyethylene glycol of the formula,
--O--(CH2CH2O)n--wherein (n) is an integer from about 10
to about 2,300.

17. The compound of claim 1, wherein R1 has an average molecular
weight from about 2,000 to about 100,000 daltons.

18. The compound of claim 1, wherein R1 has an average molecular
weight of from about 5,000 to about 60,000 daltons.

19. The compound of claim 1, wherein R1 has an average molecular
weight from about 5,000 to about 25,000 daltons or from about 20,000 to
about 45,000 daltons.

20. The compound of claim 1 wherein R2, R'2, R3 and
R'3 are independently selected from the group consisting of methyl,
ethyl and isopropyl.

21. A compound of claim 1 selected from the group consisting of:
##STR00051## ##STR00052## ##STR00053## wherein:R4 is selected from
the group consisting of OH, leaving groups, targeting groups, diagnostic
agents and biologically active moieties;(z) is a positive integer from
about 1 to about 10;(z') is zero or a positive integer from about 1 to
about 4;mPEG has the formula:
CH3--O--(CH2CH2O)n--;PEG has the formula
--O(CH2CH2O)n--; and(n) is a positive integer from about
10 to about 2,300.

22. A compound of claim 1 selected from the group consisting of:
##STR00054## ##STR00055## ##STR00056## ##STR00057## ##STR00058##
##STR00059## ##STR00060## ##STR00061## ##STR00062## ##STR00063##
##STR00064## wherein:drug is pharmaceutically active compounds, enzymes,
proteins, antibodies, monoclonal antibodies, single chain antibodies and
peptides;(z) is a positive integer from about 1 to about 10;(z') is zero
or a positive integer from about 1 to about 4;mPEG has the formula:
CH3--O(CH2CH2O)n--;PEG has the formula
--O(CH2CH2O)n--; and(n) is a positive integer from about
11 to about 2,300.

23. A method of preparing a hindered acyl or ester moiety-containing
polymeric conjugate comprising:reacting a compound of Formula
(III):A1-R1-M1 (III)with a compound of Formula (IV)
##STR00065## under conditions sufficient to form a compound of Formula
(V): ##STR00066## wherein:A1 is a capping group or M1;A2
is a capping group or ##STR00067## R1 is a substantially
non-antigenic water-soluble polymer;M1 is a leaving group;M2 is
--OH, SH or --NHR101;R100 is selected from the group consisting
of OH, or OR101;L1 and L2 are independently selected
spacers having a free electron pair positioned four to ten atoms from
C(═Y1) or C(═Y'1);Y1 and Y'1 are
independently O, S, or NR5; andR2, R3, R5, and
R100 are independently selected from the group consisting of
hydrogen, C1-6 alkyl, C2-4 alkenyl, C2-6 alkynyl,
C3-19 branched alkyl, C3-8 cycloalkyl, C1-6 substituted
alkyl, C2-6 substituted alkenyl, C2-6 substituted alkynyl,
C3-8 substituted cycloalkyl, aryl, substituted aryl, heteroaryl,
substituted heteroaryl, C1-6 heteroalkyl, substituted C1-6
heteroalkyl, C1-6 alkoxy, aryloxy, C1-6 heteroalkoxy,
heteroaryloxy, C2-6 alkanoyl, arylcarbonyl, C2-6
alkoxycarbonyl, aryloxycarbonyl, C2-6 alkanoyloxy, arylcarbonyloxy,
C2-6 substituted alkanoyl, substituted arylcarbonyl, C2-6
substituted alkanoyloxy, substituted aryloxycarbonyl, C2-6
substituted alkanoyloxy and substituted arylcarbonyloxy, or R2
together with R3 and R'2 together with R'3 independently
form a substituted or unsubstituted non-aromatic cyclohydrocarbon
containing at least three carbons;(p) is zero or a positive
integer;provided that R3 is a substituted or unsubstituted
hydrocarbon having at least tree carbons when R2 is H, and further
provided that L1 is not the same as C(R2)(R3).

24. The method of claim 23 further comprising:activating and reacting the
compound of Formula (V): ##STR00068## with a --OH or --SH containing
moiety:under conditions sufficient to form a compound of Formula (Ia):
##STR00069## wherein:A3 is a capping group or ##STR00070## R103
is selected from the group consisting of targeting agents, diagnostic
agents and biologically active moieties; andall other variables are the
same as defined in claim 24.

25. A method of treating a mammal, comprising administering an effective
amount of a compound of Formula (I) when conjugated with a biologically
active moiety to a patient in need thereof.

Description:

CROSS-REFERENCE TO RELATED APPLICATION

[0001]This application claims the benefit of priority from U.S.
Provisional Patent Application No. 60/844,942 filed Sep. 15, 2006, the
contents of which are incorporated herein by reference.

[0003]Over the years, numerous methods have been proposed for delivering
therapeutic agents into body and improving bioavailability of those
medicinal agents. One of the attempts is to include such medicinal agents
as part of a soluble transport system. Such transport systems can include
permanent conjugate-based systems or prodrugs. In particular, polymeric
transport systems can improve the solubility and stability of medicinal
agents. For example, the conjugation of water-soluble polyalkylene oxides
with therapeutic moieties such as proteins and polypeptides is known.
See, for example, U.S. Pat. No. 4,179,337 (the '337 patent), the
disclosure of which is incorporated herein by reference. The '337 patent
discloses that physiologically active polypeptides modified with PEG
circulate for extended periods in vivo, and have reduced immunogenicity
and antigenicity.

[0005]More recently, PEG has been proposed for conjugation with
oligonucleotides, especially oligonucleotides that are complementary to a
specific target messenger RNA (mRNA) sequence. Generally, nucleic acid
sequences complementary to the products of gene transcription (e.g.,
mRNA) are designated "antisense", and nucleic acid sequences having the
same sequence as the transcript or being produced as the transcript are
designated "sense". See, e.g., Crooke, 1992, Annu. Rev. Pharmacol.
Toxicol., 32: 329-376. An antisense oligonucleotide can be selected to
hybridize to all or part of a gene, in such a way as to modulate
expression of the gate. Transcription factors interact with
double-stranded DNA during regulation of transcription.

[0006]Oligonucleotides have also found use in among others, diagnostic
tests, research reagents e.g. primers in PCR technology and other
laboratory procedures. Oligonucleotides can be custom synthesized to
contain properties that are tailored to fit a desired use. Thus numerous
chemical modifications have been introduced into oligomeric compounds to
increase their usefulness in diagnostics, as research reagents and as
therapeutic entities.

[0007]Although oligonucleotides, especially antisense oligonucleotides
show promise as therapeutic agents, they are very susceptible to
nucleases and can be rapidly degraded before and after they enter the
target cells making unmodified antisense oligonucleotides unsuitable for
use in in vivo systems. Because the enzymes responsible for the
degradation are found in most tissues, modifications to the
oligonucleotides have been made in an attempt to stable the compounds and
remedy this problem. The most widely tested modifications have been made
to the back bone portion of the oligonucleotide compounds. See generally
Uhlmann and Peymann, 1990, Chemical Reviews 90, at pages 545-561 and
references cited therein. Among the many different back bones made, only
phosphorothioate showed significant antisense activity. See for example,
Padmapriya and Agrawal, 1993, Bioorg. & Med. Chem. Lett. 3, 761. While
the introduction of sulfur atoms to the back bone slows the enzyme
degradation rate, it also increases toxicity at the same time. Another
disadvantage of adding sulfur atoms is that it changes the back bone from
achiral to chiral and results in 2n diastereomers. This may cause
further side effects. Still more disadvantages of present antisense
oligonucleotides are that they may carry a negative charge on the
phosphate group which inhibits its ability to pass through the mainly
lipophilic cell membrane. The longer the compound remains outside the
cell, the more degraded it becomes resulting in less active compound
arriving at the target. A further disadvantage of present antisense
compounds is that oligonucleotides tend to form secondary and high-order
solution structures. Once these structures are formed, they become
targets of various enzymes, proteins, RNA, and DNA for binding. This
results in nonspecific side effects and reduced amounts of active
compound binding to mRNA. Other attempts to improve oligonucleotide
therapy have included adding a linking moiety and polyethylene glycol.
See for example, Kawaguchi, et al., Stability, Specific Binding Activity,
and Plasma Concentration in Mice of an Oligodeoxynucleotide Modified at
5'-Terminal with Poly(ethylene glycol), Biol. Pharm. Bull., 18(3) 474-476
(1995), and U.S. Pat. No. 4,904,582. In both of these examples, the
modifications involve the use of linking moieties that are permanent in
nature in an effort to stabilize the oligonucleotide against degradation
and increase cell permeability. However, both of these efforts fail to
provide any in vivo efficacy.

[0008]To conjugate therapeutic agents such as small molecules and
oligonucleotides to polyalkylene oxides, the hydroxyl end-groups of the
polymer must first be converted into reactive function groups. This
process is frequently referred to as "activation" and the product is
called an "activated polyalkylene oxide". Other polymers are similarly
activated.

[0009]In spite of the attempts and advances, further improvements in PEG
and polymer conjugation technology such as activated polymers have
therefore been sought. The present invention addresses this need and
others.

SUMMARY OF THE INVENTION

[0010]In order to overcome the above problems and improve the technology
for drug delivery, there are provided new activated polymers and
conjugates made therewith.

[0011]In one aspect of the present invention, there are provided compounds
of Formula (I):

##STR00001##

[0012]wherein:

[0013]A is a capping group or

##STR00002##

[0014]R1 is a substantially non-antigenic water-soluble polymer;

[0015]L1 and L'1 are independently selected spacers having a
free electron pair positioned four to ten atoms from C(═Y1) or
C(═Y'1), preferably 4 to 8 and most preferably 4 to 5 atoms from
C(═Y1) or C(═Y'1);

[0019]R4 and R'4 are independently selected from among OH,
leaving groups, targeting groups, diagnostic agents and biologically
active moieties; and

[0020](p) and (p') are independently zero or a positive integer,
preferably zero or an integer from about 1 to about 3, more preferably
zero or 1,

[0021]provided that R3 is a substituted or unsubstituted hydrocarbon
having at least three carbons when R2 is H, and further provided
that L1 is not the same as C(R2)(R3).

[0022]In certain preferred embodiments of this aspect of the invention,
the substantially non-antigenic polymer is a polyalkylene oxide and is
more preferably polyethylene glycol (hereinafter PEG). In other aspects,
the PEG is either capped on one terminal with a CH3 group, i.e.
mPEG, while in other embodiments, bis-activated PEGs are provided such as
those corresponding to the formula:

##STR00003##

[0023]Still flier aspects of the invention include methods of making the
activated polymers containing the hindered ester, methods of making
conjugates, including oligonucleotide conjugates, containing the same as
well as methods of treatment based on administering effective amounts of
conjugates containing a biologically active moiety to a patient (mammal)
in need thereof.

[0024]The polymeric delivery systems described herein include novel
linkers which can form a releasable bond such as an ester bond between
the polymer and biologically active moiety. The polymeric systems are
based on a hindered acid structure which is built as part of the polymer,
i.e. PEG backbone, and activated as an acid ester such as NHS ester on
the polymer. The activated forms can react with an hydroxy or thiol group
containing moiety to form a releasable ester bond.

[0025]One advantage of the hindered ester-based polymeric transport
systems described herein is that the polymeric delivery systems have
improved stability. Without being bound by any theories, the ester bond
in a sterically hindered environment between the polymer and a moiety
such as a leaving group, a biologically active moiety and a targeting
group can inhibit the ester linkage from being exposed to basic aqueous
medium or enzymes, and thereby stabilizes the covalent linkage. The
stability of the polymeric systems allows long-term storage prior to
attaching to targeting groups or biologically active moieties, and longer
shelf life for the polymeric conjugate containing biologically active
moieties. The improved stability increases cost efficiency.

[0026]Still further aspects of the invention include methods of making the
activated polymers containing the hindered ester, methods of making
conjugates, including oligonucleotide conjugates, containing the same as
well as methods of treatment based on administering effective amounts of
conjugates containing a biologically active moiety to a patient (mammal)
in need thereof.

[0027]Another advantage of the activated polymers corresponding to the
invention is that they are especially well suited for use with
oligonucleotides and related antisense or short-interfering RNA (siRNA)
compounds. The presence of the hindered ester group in proximity to the
oligonucleotide attached thereto provides improved stability and
resistance to nuclease degradation. It also helps decrease toxicity and
increase binding affinity to mRNA of oligonucleotide compounds.
Conjugates made in accordance with the invention provide a means for
protecting antisense oligonucleotide compounds against degradation,
preventing the formation of high-order structures. Moreover, the polymer
conjugates allow the artisan to deliver sufficient amounts of active
antisense oligonucleotide compounds to the target.

[0028]Another advantage, of the activated polymers containing the hindered
esters is that it allows the artisan to more easily conjugate
oligonucleotides of choice. There is no need to modify the
oligonucleotide or target moiety with the hindered ester before
PEGylation. The oligo is taken as is and PEGylated with the activated PEG
linker which contains the desired hindered ester protective group
thereon.

[0029]Yet another advantage of the activated polymers corresponding to the
invention is that they are especially well suited for use with
oligonucleotides and related antisense compounds. The presence of the
hindered ester group in proximity to the oligonucleotide attached thereto
provides improved stability and resistance to increase degradation. It
also helps decrease toxicity and increase binding affinity to mRNA of
oligonucleotide compounds. Conjugates made in accordance with the
invention provide a means for protecting antisense oligonucleotide
compounds against degradation, preventing the formation of high-order
structures. Moreover, the polymer conjugates allow the artisan to deliver
sufficient amounts of active antisense oligonucleotide compounds to the
target.

[0030]Although several aspects of is invention are described with respect
to linking with oligonucleotides, it will be appreciated by those of
ordinary skill that the activated polymers containing one or more
"hindering" groups thereon have a broad utility in the field of polymer
conjugation. The activated forms of the polymer can be used to releasably
and permanently link polymers such as PEG to proteins, peptides, enzymes,
small molecules, etc.

[0031]For purposes of the present invention, the terms "a biologically
active moiety" and "a residue of a biologically active moiety" shall be
understood to mean that portion of a biologically active compound which
remains after the biologically active compound has undergone a
substitution reaction in which the transport carrier portion has been
attached.

[0032]Unless otherwise defined, for purposes of the present invention:

[0036]the term "substituted cycloalkyls" include moieties such as
4-chlorocyclohexyl; aryls include moieties such as napthyl; substituted
aryls include moieties such as 3-bromophenyl; aralkyls include moieties
such as toluoyl; heteroalkyls include moieties such as ethylthiophene;

[0037]the term "substituted heteroalkyls" include moieties such as
3-methoxy-thiophene; alkoxy includes moieties such as methoxy; and
phenoxy includes moieties such as 3-nitrophenoxy;

[0038]the term "halo" shall be understood to include fluoro, chloro, iodo
and bromo; and

[0039]the terms "sufficient amounts" and "effective amounts" for purposes
of the present invention shall mean an amount which achieves a
therapeutic effect as such effect is understood by those of ordinary skin
in the art.

[0045]In most aspects of the invention, the polymers included herein are
generally described as substantially non-antigenic polymers. Within this
genus of polymers, polyalkylene oxides are preferred and polyethylene
glycols (PEG's) are most preferred. For purposes of ease of description
rather than limitation, the invention is sometimes described using PEG as
the prototypical polymer. It should be understood, however, that the
scope of the invention is applicable to a wide variety of polymers which
can be linear, substantially linear, branched, etc. One of the only
requirements is that the polymer contains the means for covalently
attaching the desired hindered ester groups described herein and that it
can withstand the processing required to transform the resulting
intermediate into the activated linker containing polymer and resulting
conjugate under the conditions described herein.

[0046]In accordance with the foregoing, there are provided compounds of
Formula (I):

##STR00004##

[0047]wherein:

[0048]A is a capping group or

##STR00005##

[0049]R1 is a substantially non-antigenic water-soluble polymer;

[0050]L1 and L'1 are independently selected spacers having a
free electron pair positioned four to ten atoms from C(═Y1) or
C(═Y'1), preferably from about 4 to about 8 and most preferably
from about 4 to about 5 atoms from C(═Y1) or C(═Y'1);

[0054]R4 and R'4 are independently selected from among OH,
leaving groups, targeting groups, diagnostic agents and biologically
active moieties; and

[0055](p) and (p') are independently zero or a positive integer,
preferably zero or an integer from about 1 to about 3, more preferably
zero or 1;

[0056]provided that R3 is a substituted or unsubstituted hydrocarbon
having at least three carbons when R2 is H, and further provided
that L1 is not the same as C(R2)(R3).

[0057]In certain preferred embodiments of this aspect of the invention,
substantially non-antigenic polymer is a polyalkylene oxide and is more
preferably polyethylene glycol (hereinafter PEG). In other aspects, the
PEG is either capped on one terminal with a CH3 group, i.e. mPEG.
Oater optional capping groups include H, NH2, OH, CO2H,
C1-6 alkoxy and C1-6 alkyl. Preferred capping groups include
methoxy and methyl.

[0058]In other embodiments, bis-activated PEGs are provided such as those
corresponding to Formula (II):

[0060]Preferably, L1 and L'1 are independently selected spacers
having a free electron pair positioned four to eight atoms from
C(═Y1) or C(--Y'1); more preferably four to six; and both Y
and Y'1 are O.

[0061]In another aspect of the invention, the biological moieties include
--OH containing moieties and --SH containing moieties.

[0062]In yet another aspect, A can be selected from among H, NH2, OH,
CO2H, C1-6 alkoxy, and C1-6 alkyls. In some preferred
embodiments, A can be methyl ethyl, methoxy, ethoxy, H, and OH. A is more
preferably methyl or methoxy.

B. Substantially Non-Antigenic Water-Soluble Polymers

[0063]Polymers employed in the polymeric delivery systems described herein
are preferably water soluble polymers and substantially non-antigenic
such as polyalkylene oxides (PAO's).

[0064]In one aspect of the invention, the compounds described herein
include a linear, terminally branched or multi-armed polyalkylene oxide.
In some preferred embodiments, the polyalkylene oxide includes
polyethylene glycol and polypropylene glycol.

[0065]The polyalkylene oxide has an average molecular weight from about
2,000 to about 100,000 daltons, preferably from about 5,000 to about
60,000 daltons. In some aspects the polyalkylene oxide can be from about
5,000 to about 25,000, and preferably from about 12,000 to about 20,000
daltons when proteins or oligonucleotides are attached or alternatively
from about 20,000 to about 45,000 daltons, and preferably from about
30,000 to about 40,000 daltons when pharmaceutically active compounds
(small molecules) are employed in the compounds described herein.

[0066]The polyalkylene oxide includes polyethylene glycols and
polypropylene glycols. More preferably, the polyalkylene oxide includes
polyethylene glycol (PEG). PEG is generally represented by the structure:

--O--(CH2CH2O)n--

where (n) is an integer from about 10 to about 2,300, and is dependent on
the number of polymer arms when multi-arm polymers are used.
Alternatively, the polyethylene glycol (PEG) residue portion of the
invention can be selected from among:

[0070]R71-74 are independently the same moieties which can be used
for R2;

[0071](a71), (a72), and (b71) are independently zero or a positive
integer, preferably 0-6, and more preferably 1; and

[0072](n) is an integer from about 10 to about 2300.

[0073]Branched or U-PEG derivatives are described in U.S. Pat. Nos.
5,643,575, 5,919,455, 6,113,906 and 6,566,506, the disclosure of each of
which is incorporated herein by reference. A non-limiting list of such
polymers corresponds to polymer systems (i)-(vii) with the following
structures:

##STR00007##

[0074]wherein:

[0075]Y61-62 are independently O, S or NR61;

[0076]Y63 is O, NR62, S, SO or SO2

[0077](w62), (w63) and (w64) are independently 0 or a positive integer;

[0078](w61) is 0 or 1;

[0079]mPEG is methoxy PEG [0080]wherein PEG is previously defined a and
a total molecular weight of the polymer portion is from about 2,000 to
about 100,000 daltons; and

[0082]In yet another aspect, the polymers include multi-arm PEG-OH or
"star-PEG" products such as those described in NOF Corp. Drug Delivery
System catalog, Ver. 8, April 2006, the disclosure of which is
incorporated herein by reference. The multi-arm polymer conjugates
contain four or more polymer arms and preferably four or eight polymer
arms.

[0083]For purposes of illustration and not limitation, the multi-arm
polyethylene glycol (PEG) residue can be

##STR00008##

[0084]wherein:

[0085](x) is 0 and a positive integer, i.e. from about 0 to about 28; and

[0086](n) is the degree of polymerization.

[0087]In one particular embodiment of the present invention, the multi-arm
PEG has the structure:

##STR00009##

wherein n is a positive integer. In one preferred embodiment of the
invention, the polymers have a total molecular weight of from about 5,000
Da to about 60,000 Da, and preferably from 12,000 Da to 40,000 Da.

[0088]In yet another particular embodiment, the multi-arm PEG has the
structure:

##STR00010##

wherein n is a positive integer. In one preferred embodiment of the
invention, the degree of polymerization for the multi-arm polymer (n) is
from about 28 to about 350 to provide polymers having a total molecular
weight of from about 5,000 Da to about 60,000 Da, and preferably from
about 65 to about 270 to provide polymers having a total molecular weight
of from 12,000 Da to 45,000 Da. This represents the number of repeating
units in the polymer chain and is dependent on the molecular weight of
the polymer.

[0089]The polymers can be converted into a suitably activated polymer,
using the activation techniques described in U.S. Pat. No. 5,122,614 or
5,808,096 patents. Specifically, such PEG can be of the formula:

##STR00011##

[0090]wherein:

[0091](u') is an integer from about 4 to about 455; and up to 3 terminal
portions of the residue is/are capped with a methyl or other lower alkyl.

[0092]In some preferred embodiments, all four of the PEG arms can be
converted to suitable activating groups, for facilitating attachment to
aromatic groups. Such compounds prior to conversion include:

##STR00012## ##STR00013##

[0093]The polymeric substances included herein are preferably
water-soluble at room temperature. A non-limiting list of such polymers
include polyalkylene oxide homopolymers such as polyethylene glycol (PEG)
or polypropylene glycols, polyoxyethylenated polyols, copolymers thereof
and block copolymers thereof, provided that the water solubility of the
block copolymers is maintained.

[0094]In a further embodiment and as an alternative to PAO-based polymers,
one or more effectively non-antigenic materials such as dextran,
polyvinyl alcohols, carbohydrate-based polymers,
hydroxypropylmethacrylamide (HPMA), polyalkene oxides, and/or copolymers
thereof can be used. See also commonly-assigned U.S. Pat. No. 6,153,655,
the contents of which are incorporated herein by reference. It will be
understood by those of ordinary skill that the same type of activation is
employed as described herein as for PAO's such as PEG. Those of ordinary
skill in the art will further realize that the foregoing list is merely
illustrative and that all polymeric materials having the qualities
described herein are contemplated. For purposes of the present invention,
"substantially or effectively non-antigenic" means all materials
understood in the art as being nontoxic and not eliciting an appreciable
immunogenic response in mammals.

[0095]In some aspects, polymers having terminal amine groups can be
employed to make the compounds described herein. The methods of preparing
polymers containing terminal amines in high purity are described in U.S.
patent application Ser. Nos. 11/508,507 and 11/537,172, the contents of
each of which are incorporated by reference. For example, polymers having
azides react with phosphine-based reducing agent such as
triphenylphosphine or an alkali metal borohydride reducing agent such as
NaBH4. Alternatively, polymers including leaving groups react with
protected amine salts such as potassium salt of methyl-tert-butyl
imidodicarbonate (KNMeBoc) or the potassium salt of di-tert-butyl
imidodicarbonate (KNBoc2) followed by deprotecting the protected
amine group. The purity of the polymers containing the terminal amines
formed by these processes is greater than about 95% and preferably
greater than 99%.

[0096]In alternative aspects, polymers having terminal carboxylic acid
groups can be employed in the polymeric delivery systems described
herein. Methods of preparing polymers having terminal carboxylic acids in
high purity axe described in U.S. patent application Ser. No. 11/328,662,
the contents of which are incorporated herein by reference. The methods
include first preparing a tertiary alkyl ester of a polyalkylene oxide
followed by conversion to the carboxylic acid derivative thereof. The
first step of the preparation of the PAO carboxylic acids of the process
includes forming an intermediate such as t-butyl ester of polyalkylene
oxide carboxylic acid. This intermediate is formed by reacting a PAO with
a t-butyl haloacetate in the presence of a base such as potassium
t-butoxide. Once the t-butyl ester intermediate has been formed, the
carboxylic acid derivative of the polyalkylene oxide can be readily
provided in purities exceeding 92%, preferably exceeding 97%, more
preferably exceeding 99% and most preferably exceeding 99.5% purity.

C. Hindered Esters

[0097]For purposes of the present invention, "hindered" shall be
understood to mean or include a sterically crowded environment around the
C(═Y1). Such environment can be made typically by including bulk
substituents, such as cyclic or branched moieties. Each of the
CR2R3 and CR'2R'3 moieties adjacent to
C(═Y1) and C(═Y'1) according to Formula (I) form
hindered esters. The R2, R'2, R3, R'3 and R5 can
be selected from among hydrogen C1-6 alkyl, C2-6 alkenyl
C2-6 alkynyl, C3-19 branched alkyl, C3-8 cycloalkyl,
C1-6 substituted alkyl, C2-6 substituted alkenyl, C2-6
substituted alkynyl, C3-8 substituted cycloalkyl, aryl, substituted
aryl, heteroaryl, substituted heteroaryl, C1-6 heteroalkyl,
substituted C1-6 heteroalkyl, C1-6 alkoxy, aryloxy, C1-6
heteroalkoxy, heteroaryloxy, C2-6 alkanoyl, arylcarbonyl, C2-6
alkoxycarbonyl, aryloxycarbonyl, C2-6 alkanoyloxy, arylcarbonyloxy,
C2-6 substituted alkanoyl, substituted arylcarbonyl, C2-6
substituted alkanoyloxy, substituted aryloxycarbonyl, C2-6
substituted alkanoyloxy and substituted arylcarbonyloxy. Any of the
possible groups described herein for R2 and R3 (R'2 and
R'3) can be used so long as both R2 and R3 (R'2 and
R'3) are not simultaneously H. When one of R2 and R3
(R'2 and R'3) is H, the other contains at least three
hydrocarbons.

[0099]In an alternative embodiment, R2 together with R3 and
R'2 together with R'3 can form a substituted or unsubstituted
non-aromatic cyclohydrocarbon containing at least three carbons.

D. Spacers

L1 and L'1

[0100]In another aspect of the present invention, free electron pairs of
the L1 and L'1 spacers linked to the CR2R3 and
CR'2R'3 moieties provide enchimeric effects. Without being
bound by any theory, the free electron pairs positioned four to ten atoms
from C(═Y1) and C(═Y'1) facilitate (modify) release
rate of biologically active moieties, target groups and diagnostic agents
from the polymeric delivery systems described herein.

[0101]In one preferred embodiment, the L1 and L'1 spacers can be
selected from among:

[0104](s) and (s') are independently zero or a positive integer;
preferably from about 1 to about 4; and

[0105](r) is 0 or 1.

[0106]Alternatively, the L1 and L'1, groups can be selected from
among:

--NH--(CH2--CH2O)p--CH12--,
--C(═O)--(CH2)n--, --NH--(CH2)n--,

--S--(CH2)p--,

--NH--(CH2)p--O--CH2-- and

--NH--C(═O)--(CH2)p--NH--C(═O)--(CH2)q--

[0107]wherein

[0108](p) is an integer from 1 to 12; and

[0109](n) and q are independently a positive integer, preferably from
about 1 to 8, and more preferably from about 1 to 4.

[0110]In yet another preferred embodiment, the free electron pairs of the
L1-2 and L'1-2 spacers are positioned four to eight atoms from
C(═Y1) and C(═Y'1). More preferably, the electron pairs
are positioned four to five atoms from C(═Y1) and
C(═Y'1).

[0111]Preferred embodiments according to the preferred aspect are
-L1-C(R2)(R3)--C(═Y1)-- and
-L'1-C(R'2)(R'3)--C(═Y'1) include:

##STR00014##

[0112]In another aspect, the polymeric delivery systems described herein
include that R3 is a substituted or unsubstituted hydrocarbon having
at least three carbons when R2 is H, and L1 is not the same as
C(R2)(R3).

[0117](t) and (t') are independently zero or a positive integer,
preferably zero or an integer from about 1 to about 12, more preferably
an integer from about 1 to about 8, and most preferably 1 or 2; and

[0118](v) and (v') are independently zero or 1.

[0119]In a preferred embodiment L2 and L'2 can be selected from
among:

[0131]For purposes of the present invention, leaving groups are to be
understood as those groups which are capable of reacting with a
nucleophile found on the desired target, i.e. a biologically active
moiety, a diagnostic agent, a targeting moiety, a bifunctional spacer,
intermediate, etc. The targets thus contain a group for displacement,
such as OH or SH groups found on proteins, peptides, enzymes, naturally
or chemically synthesized therapeutic molecules such as doxorubicin, and
spacers such as mono-protected diamines.

[0136]In one aspect of the invention, the biologically active compounds
are suitable for medicinal or diagnostic use in the treatment of animals,
e.g., mammals, including humans, for conditions for which such treatment
is desired.

[0137]In yet another aspect, hydroxyl- or thiol-containing biologically
active moieties are within the scope of the present invention. The only
limitations on the types of the biologically active moieties suitable for
inclusion herein is that there is available at least one hydroxyl- or
thiol-group which can react and link with a carrier portion and that
there is not substantial loss of bioactivity in the form of conjugated to
the polymeric delivery systems described herein.

[0138]Alternatively, parent compounds suitable for incorporation into the
polymeric transport conjugate compounds of the invention, may be active
after hydrolytic release from the linked compound, or not active after
hydrolytic release but which will become active after undergoing a
further chemical process/reaction. For example, an anticancer drug that
is delivered to the bloodstream by the polymeric transport system, may
remain inactive until entering a cancer or tumor cell, whereupon it is
activated by the cancer or tumor cell chemistry, e.g., by an enzymatic
reaction unique to that cell.

[0140]For purposes of the present invention it shall be understood to mean
that the pharmaceutically active compounds include small molecular weight
molecules. Typically, pharmaceutically active compounds have a molecular
weight of less than about 1,500 daltons. A non-limiting list of such
compounds includes camptothecin and analogs such as SN38 or irinotecan
hydroxyl- or thiol-topoisomerase I inhibitors, taxanes and paclitaxel
derivatives, nucleosides including AZT and acyclovir, anthracycline
compounds including daunorubicin and doxorubicin, related anti-metabolite
compounds including Ara-C (cytosine arabinoside) and gemcitabine, etc.

[0141]In another preferred embodiment, the choice for conjugation is an
oligonucleotide and after conjugation, the target is referred to as a
residue of an oligonucleotide. The oligonucleotides can be selected from
among any of the known oligonucleotides and oligodeoxynucleotides with
phosphorodiester backbones or phosphorothioate backbones, locked nucleic
acid (LNA), nucleic acid with peptide backbone (PNA), tricyclo-DNA,
double stranded oligonucleotide (decoy ODN), catalytic RNA sequence
(RNAi), ribozymes, spiegelmers, and CpG oligomers.

[0142]The "polynucleotide" (or "oligonucleotide") of the above group of
compound includes oligonucleotides and oligodeoxynucleotides, including,
for example, an oligonucletide that has the same or substantially similar
nucleotide sequence as does Genasense (a/k/a oblimersen sodium, produced
by Genta Inc., Berkeley Heights, N.J.). Genasense is an 18-mer
phosphorothioate antisense oligonucleotide, TCTCCCAGCGTGCGCCAT (SEQ ID
NO: 1), that is complementary to the first six codons of the initiating
sequence of the human bcl-2 mRNA (human bcl-2 mRNA is art-known, and is
described, e.g., as SEQ ID NO: 19 in U.S. Pat. No. 6,414,134,
incorporated by reference herein). The U.S. Food and Drug Administration
(FDA) gave Genasense Orphan Drug status in August 2000.

[0143]Further, oligonucleotides and oligodeoxynucleotides useful according
to the invention include, but are not limited to, the following:

[0144]Oligonucleotides and oligodeoxynucleotides with natural
phosphorodiester backbone or phosphorothioate backbone or any other
modified backbone analogues;

[0156]Although antisense oligonucleotides and related compounds have been
mentioned as preferred targets for the attachment of the polymers
containing the hindered esters, it is intended that R4 or R'4
include all suitable biologically active proteins, peptides, enzymes,
small molecules etc. known to benefit from PEG or polymer attachment.

[0157]Modifications to the oligonucleotides contemplated in the invention
include, for example, the addition to or substitution of selected
nucleotides with functional groups or moieties that permit covalent
linkage of an oligonucleotide to a desirable polymer, and/or the addition
or substitution of functional moieties that incorporate additional
charge, polarizability, hydrogen bonding, electrostatic interaction, and
functionality to an oligonucleotide. Such modifications include, but are
not limited to, 2'-position sugar modifications, 5-position pyrimidine
modifications, 8-position purine modifications, modifications at
exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo
or 5-iodouracil, backbone modifications, methylations, base-pairing
combinations such as the isobases isocytidine and isoguanidine, and
analogous combinations. Oligonucleotide modifications can also include 3'
and 5' modifications such as capping.

[0158]Structures of illustrative nucleoside analogs are provided below.
See more examples of nucleoside analogues described in Freier & Altman;
Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug
Development, 2000, 3(2), 293-213, the contents of each of which axe
incorporated herein by reference.

[0160]A further aspect of the invention provides the conjugate compounds
optionally prepared with a diagnostic tag linked to the polymeric
delivery system described herein, wherein the tag is selected for
diagnostic or imaging purposes. Thus, a suitable tag is prepared by
linking any suitable moiety, e.g., an amino acid residue, to any
art-standard emitting isotope, radio-opaque label, magnetic resonance
label, or other non-radioactive isotopic labels suitable for magnetic
resonance imaging, fluorescence-type labels, labels exhibiting visible
colors and/or capable of fluorescing under ultraviolet, infrared or
electrochemical stimulation, to allow for imaging tumor tissue during
surgical procedures, and so forth. Optionally, the diagnostic tag is
incorporated into and/or linked to a conjugated therapeutic moiety,
allowing for monitoring of the distribution of a therapeutic biologically
active material within an animal or human patient.

[0161]In yet a further aspect of the invention, the inventive tagged
conjugates are readily prepared, by art-known methods, with any suitable
label, including, e.g., radioisotope labels. Simply by way of example,
these include 131Iodine, 125Iodine, 99m-Technetium and/or
111Indium to produce radioimmunoscintigraphic agents for selective
uptake into tumor cells, in vivo. For instance, there are a number of
art-known methods of linking peptide to Tc-99m including, simply by way
of example, those shown by U.S. Pat. Nos. 5,328,679; 5,888,474;
5,997,844; and 5,997,845, incorporated by reference herein.

G. Preferred Embodiments Corresponding to Formula

[0162]Some particular embodiments prepared by the methods described herein
include:

G. Methods of Making the Activated Polymers and Conjugates Made Therewith

[0174]In one aspect of the invention, the polymeric compound having
hindered ester can be prepared by conjugating a polymeric compound having
a OH or a leaving group at the terminal end with a nucleophile having a
protected hindered ester or a hindered acid at the distal end. Further
deprotecting and activating the resulting polymeric compound will provide
the compound of the current invention. The terminal group of the current
invention can be either carboxylic acid form ready to be coupled with a
OH or SH containing moiety or an activated form which can be replaced
upon conjugating with OH or SH containing moiety.

[0175]Alternatively, a OH or SH containing compound can be conjugated to
form a hindered ester intermediate, which in turn reacted with an
activated polymer for the polymeric conjugate having a hindered ester
with a biologically active moiety.

[0183]M1 is a leaving group such as halogens, activated carbonates,
isocyanate, N-hydroxysuccinimidyl, tosylate, mesylate, tresylate,
nosylate, ortho-nitrophenoxy, imidazole and other leaving groups known by
those of ordinary skill in the art;

[0187]The attachment of the hindered ester moiety according to Formula
(IV) to the PEG or other polymer can be done using standard chemical
synthetic techniques well known to those of ordinary skill. The activated
polymer portion such as SC-PEG, PEG-amine, PEG acids, etc. can be
obtained from either commercial sources or synthesized by the artisan
without undue experimentation.

[0188]For the purpose of the current invention, a non-limiting list of
such hindered ester moiety includes:

##STR00038##

[0189]The compounds of Formula (V) can further react with a --OH or --SH
containing moiety in the presence of base and a coupling agent under
conditions sufficient to form a compound of Formula (Ia):

##STR00039##

[0190]wherein:

[0191]A3 is a capping group or

##STR00040##

[0192]R103 is selected from among targeting agents, diagnostic agents
and biologically active moieties; and all other variables are previously
defined.

[0193]For purposes of the present invention, the R103 is shall be
understood as the portion of the OH or SH containing moiety which remains
after it has undergone a reaction with the compound of Formula (V).

[0194]Alternatively, the compounds described herein can be prepared by
methods including:

[0195]reacting a compound of Formula (VI):

##STR00041##

[0196]with a compound of Formula (VII):

A4-R1-M4 (VII)

[0197]under conditions sufficient to form a compound of Formula (VIII):

##STR00042##

[0198]wherein

[0199]A4 is a capping group or M4;

[0200]A5 is a capping group or

##STR00043##

[0201]M3 is --OH, SH, or --NHR105;

[0202]M4 is a leaving group such as halogens, activated carbonates,
isocyanate, N-hydroxysuccinimidyl, tosylate, mesylate, tresylate,
nosylate, ortho-nitrophenoxy, imidazole and other leaving groups known by
those of ordinary skill in the art;

[0206]Attachment of the hindered ester containing group to the polymer
portion is preferably carried out in the presence of a coupling agent A
non-limiting list of suitable coupling agents include
1,3-diisopropylcarbodiimide (DIPC), any suitable dialkyl carbodiimides,
2-halo-1-alkyl-pyridinium halides, (Mukaiyama reagents),
1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC), propane phosphonic
acid cyclic anhydride (PPACA) and phenyl dichlorophosphates, etc. which
are available, for example from commercial sources such as Sigma-Aldrich
Chemical, or synthesized using known techniques.

[0207]Preferably, the reactions are carried out in an inert solvent such
as methylene chloride, chloroform, DMF or mixtures thereof. The reactions
can be preferably conducted in the presence of a base, such as
dimethylaminopyridine (DMAP), diisopropylethylamine, pyridine,
triethylamine, etc. to neutralize any acids generated. The reactions can
be cared out at a temperature from about 0° C. up to about
22° C. (room temperature).

H. Methods of Treatment

[0208]Another aspect of the present invention provides methods of
treatment for various medical conditions in mammals. The methods include
administering, to the mammal in need of such treatment, an effective
amount of a compound described herein when conjugated to a biologically
active moiety. The polymeric conjugate compounds are useful for, among
other things, treating diseases which are similar to those which are
treated with the parent compound, e.g. enzyme replacement therapy,
neoplastic disease, reducing tumor burden, preventing metastasis of
neoplasms and preventing recurrences of tumor/neoplastic growths in
mammals.

[0209]The amount of the polymeric conjugate that is administered will
depend upon the amount of the parent molecule included therein.
Generally, the amount of polymeric conjugate used in the treatment
methods is that amount which effectively achieves the desired therapeutic
result in mammals. Naturally, the dosages of the various polymeric
conjugate compounds will vary somewhat depending upon the parent
compound, molecular weight of the polymer, rate of in vivo hydrolysis,
etc. Those skilled in the art will determine the optimal dosing of the
polymeric transport conjugates selected based on clinical experience and
the treatment indication. Actual dosages will be apparent to the artisan
without undue experimentation.

[0210]The compounds of the present invention can be included in one or
more suitable pharmaceutical compositions for administration to mammals.
The pharmaceutical compositions may be in the form of a solution,
suspension, tablet, capsule or the like, prepared according to methods
well known in the art. It is also contemplated that administration of
such compositions may be by the oral and/or parenteral routes depending
upon the needs of the artisan. A solution and/or suspension of the
composition may be utilized, for example, as a carrier vehicle for
injection or infiltration of the composition by any art known methods,
e.g., by intravenous, intramuscular, intraperitoneal, subcutaneous
injection and the like. Such administration may also be by infusion into
a body space or cavity, as well as by inhalation and/or intranasal
routes. In preferred aspects of the invention, however, the polymeric
conjugates are parenterally administered to mammals in need thereof.

EXAMPLES

[0211]The following examples serve to provide further appreciation of the
invention but are not meant in any way to restrict the scope of the
invention. The bold-faced numbers recited in the Examples correspond to
those shown in FIG. 1-4. Abbreviations are used throughout the examples
such as, DCM (dichloromethane), DIPEA (diisopropylethylamine), DMAP
(4-dimethylaminopyridine), DMF (N,N'-dimethylformamide), EDC
(1-(3-dimethylaminopropyl)-3-ethyl carbodiimide), IPA (isopropanol), Mmt
(4-methoxytriphenylmethyl), NHS (N-hydroxysuccinimide), PEG (polyethylene
glycol), SCA-SH (single-chain antibody), SC-PEG (succinimidyl carbonate
polyethylene glycol), TEAA (tetraethylammonium acetate), TFA
(trifluoroacetic acid), and THF (tetrahydrofuran).

[0212]General Procedures. All reactions are run under an atmosphere of dry
nitrogen or argon. Commercial reagents are used without further
purification. All PEG compounds are dried under vacuum or by azeotropic
distillation from toluene prior to use. 13C NMR spectra were
obtained at 75.46 MHz using a Varian Mercury® 300 NMR spectrometer
and deuterated chloroform and pyridine as the solvents unless otherwise
specified Chemical shifts (δ) are reported in parts per minion
(ppm) downfield from tetramethylsilane (TMS).

[0213]HPLC Method. The reaction mixtures and the purity of intermediates
and final products are monitored by a Beckman Coulter System Gold®
HPLC instrument. It employs a ZORBAX® 300SB C8 reversed phase column
(150×4.6 mm) or a Phenomenex Jupiter® 300A C18 reversed phase
column. (150×4.6 mm) with a 168 Diode Array UV Detector, using a
gradient of 5-80% of acetonitrile in 0.05 M tetraethylammonium acedtate
(TFAA) at a flow rate of 1 mL/min.)

[0216]Butyllithium (1.6 M solution in t-BuOH, 200 ml) was added to a
solution of ethyl isobutyrate (compound 6, 35 g) in F (500 mL) at
-78° C. and the solution was stirred for 1 h at the same
temperature. 1,5-Dibromopetane (compound 7, 100 g) was added and the
mixture was allowed to warm up to room temperature. The mire was stirred
at room temperature for 1 hour and was poured into aqueous sodium
bicarbonate (500 mL). The organic layer was evaporated. The residue was
purified by a silica gel column, eluted with 10% ethyl acetate in hexane
to give the desired product as a liquid (29.2 g, yield 36.7%).

Example 4

Preparation of N3--HE-OEt, Compound (9)

[0217]Ethyl 7-bromo-2,2-dimethylheptanoate (compound 8, 26.5 g) was heated
with sodium azide (13 g) in DMF (500 mL) at 100° C. for 2 hours.
The mixture was concentrated and the residue was purified by a silica gel
column, eluted with 10% ethyl acetate in hexane to give the desired
product as a liquid (20.5 g, yield 90.3%).

Example 5

Preparation of N3--HE-OH, Compound (10)

[0218]Ethyl 7-azido-2,2-dimethylheptanoate (compound 9, 20.5 g) was heated
with sodium hydroxide (10 g, 85%) in ethanol (500 mL) under reflux for 2
hours. The mixture was concentrated and water (400 mL) was added. The
mixture was acidified with concentrated hydrochloric acid to pH 2 and
extracted with ethyl acetate (500 mL). The organic layer was concentrated
and the residue was purified by a silica gel column, eluted with 50%
ethyl acetate in hexane to give the desired product as a liquid (17.1 g,
yield 95%).

Example 6

Preparation of N3--HE-T, Compound (12)

[0219]7-Azido-2,2-dimethylheptanoic acid (compound 10, 8 g) was dissolved
in dichloromethane (200 mL). Oxalyl chloride (6.4 g) was added and the
mixture was refluxed for 2 h and evaporated. The residue was dissolved in
dichloromethane (100 mL) and was added in 3'-acetyl thymidine (compound
11, 5.85 g) in pyridine (100 mL). The solution was stirred at room
temperate for 24 hours and was poured into aqueous sodium bicarbonate
(500 mL). The mixture was extracted with dichloromethane (500 mL) and the
organic layer was concentrated. The residue was purified by a silica gel
column, eluted with 5% methanol in DCM to give the desired product as a
colorless solid (5.6 g, yield 61%).

Example 7

Preparation of NH2--HE-T, Compound (13)

[0220]5'-(7-Azido-2,2-dimethylheptanoyl) 3'-acetylthymidine (compound 12,
4.65 g) was hydrogenated in methanol (200 mL) under 30 psi in the
presence of Pd/C (10%, 0.5 g) for 1 h. The mixture was filtered and the
filtrate was evaporated to give a solid (4.4 g, yield 100%).

Example 8

Preparation of MmtNH--HE-T, Compound (14)

[0221]5'-(7-Amino-2,2-dimethylheptanoyl) 3'-acetylthymidine (compound 13,
4.4 g), triethylamine (4 ml) and 4-methoxytrityl chloride (7.5 g) were
stirred in pyridine (100 mL) for 10 h. Methylamine (40%, 10 mL) was added
and the solution was stirred for 2 h. The mixture was poured into aqueous
sodium bicarbonate (500 mL) and extracted with dichloromethane (500 mL).
The organic layer was concentrated. The residue was purified by a silica
gel column, eluted with 5% methanol in dichloromethane to give the
desired product as a colorless solid (4.9 g, yield 71%).

[0223]Compound 15 was transferred to Trilink Biotechnologies, CA to use as
the last monomer in the oligo synthesis. The Mint group was deprotected
after the synthesis and the oligo was purified by RP-HPLC and compound 16
as the free anine was obtained for PEG conjugation. The sequence of
oligonucleotide was TCTCCCAGCGTGCGCCAT.

Example 11

Preparation of PEG-HE-Oligo, Compounds (17)

[0224]To a solution of compound 16 (10 mg, 1.7 μmol) in PBS buffer (5
mL, pH 7.8) was added SC-PEG (compound 1, Mw 30 kDa, 520 mg, 17 μmol)
and stirred at room temperature for 5 hrs. The reaction mixture was
diluted to 50 mL with water and loaded on a Poros HQ, strong anion
exchange column (10 mm×1.5 mm, bed volume ˜16 mL) which was
pre-equilibrated with 20 mM Tris-HCl buffer, pH 7-4 (buffer A). The
column was washed with 3-4 column volumes of buffer A to remove the
excess PEG linker. Then the product was eluted with a gradient of 0 to
100% 1 M NaCl in 20 mM Tris-HCl buffer, pH 7.4, buffer B in 10 min,
followed by 100% buffer B for 10 min at a flow rate of 10 mL/min. The
eluted product was desalted using HiPrep desalting column (50 mL) and
lyophilized to give 6 mg of the product. The equivalent of
oligonucleotide in the conjugate measured by UV was 60%, wt/wt.

Example 12

Preparation of PEG-Linker-HE-Oligo Compound (19)

[0225]To a solution of compound 16 (10 mg, 1.7 mmol) in PBS buffer (5 mL,
pH 7.8) was added PEG-Linker-NHS (compound 18, Mw 30 kDa, 520 mg, 17
μmol) and stirred at room temperature for 5 hrs. The reaction mixture
was diluted to 50 mL with water and loaded on a Poros HQ, strong anion
exchange column (10 mm×1.5 mm, bed volume ˜16 mL) which was
pre-equilibrated with 20 mM Tris-HCl buffer, pH 74 (buffer A). The column
was washed with 3-4 column volumes of buffer A to remove the excess PEG
linker. Then the product was eluted with a gradient of 0 to 100% 1 M NaCl
in 20 mM Tris-HCl buffer, pH 7.4, buffer B in 10 min, followed by 100%
buffer B for 10 min at a flow rate of 10 mL/min. The eluted product was
desalted using HiPrep desalting column (50 mL) and lyophilized to solid
to give 5 mg of the desired product. The equivalent of oligonucleotide in
the conjugate measured by UV was 50%, wt/wt.

[0230]The rates of hydrolysis were obtained by employing a CS reversed
phase column (Zorbax® SB-C8) using a gradient mobile phase consisting
of (a) 0.1 M triethylammonium acetate buffer and (b) acetonitrile. A flow
rate of 1 mL/min was used, and chromatograms were monitored using a UV
detector at 227 nm for paclitaxel and 260 nm for oligonucleotides. For
hydrolysis in buffer, PEG derivatives were dissolved in 0.1 M pH 7.4 PBS
or water at a concentration of 5 mg/mL, while for hydrolysis in plasma,
the derivatives were dissolved in distilled water at a concentration of
20 mg/100 μL and 900 μL of rat plasma was added to this solution.
The mixture was vortexed for 2 min and divided into 2 mL glass vials with
100 μL of the aliquot per each vial. The solutions were incubated at
37° C. for various periods of time. A mixture of
methanol-acetonitrile (1:1 v/v, 400 μL) was added to a vial at the
proper interval and the mixture was vortexed for 1 min, followed by
filtration through 0.45 mm filter membrane (optionally followed by a
second filtration through 0.2 mm filter membrane). An aliquot of 20 μL
of the filtrate was injected into the HPLC. On the basis of the peak
area, the amounts of native compound and PEG derivative were estimated,
and the half-life of each compound in different media was calculated
using linear regression analysis from the disappearance of PEG
derivative. Table below shows the result of the stability study for
compounds in the examples.